Clark's nutcracker forest community visitation: Whitebark pine maintains a keystone seed disperser.

Clark's nutcrackers (Nucifraga columbiana) are obligate seed dispersers for whitebark pine (Pinus albicaulis), but they frequently use other conifer seed resources because of annual variability in cone production or geographic variation in whitebark pine availability. Whitebark pine is declining from several threats including white pine blister rust, leading to potential population declines in the nutcracker and the pine. We hypothesize that where there are few additional seed resources, whitebark pine becomes the key and limiting resource supporting nutcracker populations. We investigated how nutcrackers use coniferous forest community types within Yellowstone National Park to determine potential seed resources and the importance of whitebark pine. We established sites representing five forest community types, including whitebark pine, lodgepole pine (P. contorta), Engelmann spruce (Picea engelmannii), limber pine (P. flexilis), and Douglas-fir (Pseudotsuga menziesii). Each transect annually generated nutcracker point counts, conifer cone production indices, community composition data, and seed resource use observations. We compared hierarchical distance sampling models, estimating nutcracker density and its relationship to forest community type, seed harvesting time-period, year, study site, and cone seed energy. We found cone production varied across years indicating annual variability in energy availability. Nutcracker density was best predicted by forest community type and survey time-period and was highest in whitebark pine stands during the mid-harvesting season. Nutcracker density was comparatively low for all other forest community types. This finding underscores the importance of whitebark pine as a key seed resource for Clark's nutcracker in Yellowstone National Park. The decline of whitebark pine potentially leads to a downward spiral in nutcrackers and whitebark pine, arguing for continued monitoring of nutcrackers and implementation of restoration treatments for whitebark pine.

Development of nuclear microsatellite loci for Pinus albicaulis Engelm. (Pinaceae), a conifer of conservation concern.

Pinus albicaulis (whitebark pine) is a widely-distributed but rapidly declining high elevation western North American tree and a candidate for listing under the U.S. Endangered Species Act. Our objectives were to develop reliable nuclear microsatellite markers that can be used to assess within-population genetic diversity as well as seed and pollen migration dynamics, and to validate markers using two geographically proximal P. albicaulis populations. We identified 1,667 microsatellite-containing sequences from shotgun DNA libraries of P. albicaulis. Primer pairs were designed for 308 unique microsatellite-containing loci, and these were evaluated for PCR amplification success and segregation in a panel of diploid needle tissue. DNA was extracted with an SDS protocol, and primers were screened through gel electrophoresis. Microsatellites were genotyped through fluorescent primer fragment analysis. Ten novel and 13 transferred loci were found to be reproducible in analyses based on 20 foliage samples from each of two locations: Henderson Mountain, Custer Gallatin National Forest, Montana, and Mt. Washburn, Yellowstone National Park, Wyoming (USA). Transferred loci had higher numbers of alleles and expected heterozygosities than novel loci, but also revealed evidence for a higher frequency of null alleles. Eight of the 13 transferred loci deviated significantly from Hardy-Weinberg Equilibrium, and showed large positive FIS values that were likely inflated by null alleles. Mantel's tests of transferred and novel markers showed no correlation between genetic and geographic distances within or among the two sampled populations. AMOVA suggests that 91% of genetic variability occurs within populations and 9% between the two populations. Studies assessing genetic diversity using these microsatellite loci can help guide future management and restoration activities for P. albicaulis.

Microsite and elevation zone effects on seed pilferage, germination, and seedling survival during early whitebark pine recruitment.

Tree recruitment is a spatially structured process that may undergo change over time because of variation in postdispersal processes. We examined seed pilferage, seed germination, and seedling survival in whitebark pine to determine whether 1) microsite type alters the initial spatial pattern of seed caches, 2) higher abiotic stress (i.e. higher elevations) exacerbates spatial distribution changes, and 3) these postdispersal processes are spatially clustered. At two study areas, we created a seed distribution pattern by burying seed caches in microsite types frequently used by whitebark pine's avian seed disperser (Clark's nutcracker) in upper subalpine forest and at treeline, the latter characterized by high abiotic environmental stress. We monitored caches for two years for pilferage, germination, and seedling survival. Odds of pilferage (both study areas), germination (northern study area), and survival (southern study area) were higher at treeline relative to subalpine forest. At the southern study area, we found higher odds of 1) pilferage near rocks and trees relative to no object in subalpine forest, 2) germination near rocks relative to trees within both elevation zones, and 3) seedling survival near rocks and trees relative to no object at treeline. No microsite effects were detected at the northern study area. Findings indicated that the microsite distribution of seed caches changes with seed/seedling stage. Higher odds of seedling survival near rocks and trees were observed at treeline, suggesting abiotic stress may limit safe site availability, thereby shifting the spatial distribution toward protective microsites. Higher odds of pilferage at treeline, however, suggest rodents may limit treeline recruitment. Further, odds of pilferage were higher near rocks and trees relative to no object in subalpine forest but did not differ among microsites at treeline, suggesting pilferage can modulate the spatial structure of regeneration, a finding supported by limited clustering of postdispersal processes.

Whitebark pine facilitation at treeline: potential interactions for disruption by an invasive pathogen.

In stressful environments, facilitation often aids plant establishment, but invasive plant pathogens may potentially disrupt these interactions. In many treeline communities in the northern Rocky Mountains of the U.S. and Canada, Pinus albicaulis, a stress-tolerant pine, initiates tree islands at higher frequencies than other conifers - that is, leads to leeward tree establishment more frequently. The facilitation provided by a solitary (isolated) P. albicaulis leading to tree island initiation may be important for different life-history stages for leeward conifers, but it is not known which life-history stages are influenced and protection provided. However, P. albicaulis mortality from the non-native pathogen Cronartium ribicola potentially disrupts these facilitative interactions, reducing tree island initiation. In two Rocky Mountain eastern slope study areas, we experimentally examined fundamental plant-plant interactions which might facilitate tree island formation: the protection offered by P. albicaulis to leeward seed and seedling life-history stages, and to leeward krummholz conifers. In the latter case, we simulated mortality from C. ribicola for windward P. albicaulis to determine whether loss of P. albicaulis from C. ribicola impacts leeward conifers. Relative to other common solitary conifers at treeline, solitary P. albicaulis had higher abundance. More seeds germinated in leeward rock microsites than in conifer or exposed microsites, but the odds of cotyledon seedling survival during the growing season were highest in P. albicaulis microsites. Planted seedling survival was low among all microsites examined. Simulating death of windward P. albicaulis by C. ribicola reduced shoot growth of leeward trees. Loss of P. albicaulis to exotic disease may limit facilitation interactions and conifer community development at treeline and potentially impede upward movement as climate warms.

Blood from a turnip: tissue origin of low-coverage shotgun sequencing libraries affects recovery of mitogenome sequences.

Next generation sequencing methods allow rapid, economical accumulation of data that have many applications, even at relatively low levels of genome coverage. However, the utility of shotgun sequencing data sets for specific goals may vary depending on the biological nature of the samples sequenced. We show that the ability to assemble mitogenomes from three avian samples of two different tissue types varies widely. In particular, data with coverage typical of microsatellite development efforts (∼1×) from DNA extracted from avian blood failed to cover even 50% of the mitogenome, relative to at least 500-fold coverage from muscle-derived data. Researchers should consider possible applications of their data and select the tissue source for their work accordingly. Practitioners analyzing low-coverage shotgun sequencing data (including for microsatellite locus development) should consider the potential benefits of mitogenome assembly, including internal barcode verification of species identity, mitochondrial primer development, and phylogenetics.

Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies.

As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (∼3.5-5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies.

Whitebark pine stand condition, tree abundance, and cone production as predictors of visitation by Clark's nutcracker.

BACKGROUND: Accurately quantifying key interactions between species is important for developing effective recovery strategies for threatened and endangered species. Whitebark pine (Pinus albicaulis), a candidate species for listing under the Endangered Species Act, depends on Clark's nutcracker (Nucifraga columbiana) for seed dispersal. As whitebark pine succumbs to exotic disease and mountain pine beetles (Dendroctonus ponderosae), cone production declines, and nutcrackers visit stands less frequently, reducing the probability of seed dispersal. METHODOLOGY/PRINCIPAL FINDINGS: We quantified whitebark pine forest structure, health metrics, and the frequency of nutcracker occurrence in national parks within the Northern and Central Rocky Mountains in 2008 and 2009. Forest health characteristics varied between the two regions, with the northern region in overall poorer health. Using these data, we show that a previously published model consistently under-predicts the proportion of survey hours resulting in nutcracker observations at all cone density levels. We present a new statistical model of the relationship between whitebark pine cone production and the probability of Clark's nutcracker occurrence based on combining data from this study and the previous study. CONCLUSIONS/SIGNIFICANCE: Our model clarified earlier findings and suggested a lower cone production threshold value for predicting likely visitation by nutcrackers: Although nutcrackers do visit whitebark pine stands with few cones, the probability of visitation increases with increased cone production. We use information theoretics to show that beta regression is a more appropriate statistical framework for modeling the relationship between cone density and proportion of survey time resulting in nutcracker observations. We illustrate how resource managers may apply this model in the process of prioritizing areas for whitebark pine restoration.

Rapid microsatellite identification from Illumina paired-end genomic sequencing in two birds and a snake.

Identification of microsatellites, or simple sequence repeats (SSRs), can be a time-consuming and costly investment requiring enrichment, cloning, and sequencing of candidate loci. Recently, however, high throughput sequencing (with or without prior enrichment for specific SSR loci) has been utilized to identify SSR loci. The direct "Seq-to-SSR" approach has an advantage over enrichment-based strategies in that it does not require a priori selection of particular motifs, or prior knowledge of genomic SSR content. It has been more expensive per SSR locus recovered, however, particularly for genomes with few SSR loci, such as bird genomes. The longer but relatively more expensive 454 reads have been preferred over less expensive Illumina reads. Here, we use Illumina paired-end sequence data to identify potentially amplifiable SSR loci (PALs) from a snake (the Burmese python, Python molurus bivittatus), and directly compare these results to those from 454 data. We also compare the python results to results from Illumina sequencing of two bird genomes (Gunnison Sage-grouse, Centrocercus minimus, and Clark's Nutcracker, Nucifraga columbiana), which have considerably fewer SSRs than the python. We show that direct Illumina Seq-to-SSR can identify and characterize thousands of potentially amplifiable SSR loci for as little as $10 per sample--a fraction of the cost of 454 sequencing. Given that Illumina Seq-to-SSR is effective, inexpensive, and reliable even for species such as birds that have few SSR loci, it seems that there are now few situations for which prior hybridization is justifiable.

Invasive pathogen threatens bird-pine mutualism: implications for sustaining a high-elevation ecosystem.

Human-caused disruptions to seed-dispersal mutualisms increase the extinction risk for both plant and animal species. Large-seeded plants can be particularly vulnerable due to highly specialized dispersal systems and no compensatory regeneration mechanisms. Whitebark pine (Pinus albicaulis), a keystone subalpine species, obligately depends upon the Clark's Nutcracker (Nucifraga columbiana) for dispersal of its large, wingless seeds. Clark's Nutcracker, a facultative mutualist with whitebark pine, is sensitive to rates of energy gain, and emigrates from subalpine forests during periods of cone shortages. The invasive fungal pathogen Cronartium ribicola, which causes white pine blister rust, reduces whitebark pine cone production by killing cone-bearing branches and trees. Mortality from blister rust reaches 90% or higher in some whitebark pine forests in the Northern Rocky Mountains, USA, and the rust now occurs nearly rangewide in whitebark pine. Our objectives were to identify the minimum level of cone production necessary to elicit seed dispersal by nutcrackers and to determine how cone production is influenced by forest structure and health. We quantified forest conditions and ecological interactions between nutcrackers and whitebark pine in three Rocky Mountain ecosystems that differ in levels of rust infection and mortality. Both the frequency of nutcracker occurrence and probability of seed dispersal were strongly related to annual whitebark pine cone production, which had a positive linear association with live whitebark pine basal area, and negative linear association with whitebark pine tree mortality and rust infection. From our data, we estimated that a threshold level of approximately 1000 cones/ha is needed for a high likelihood of seed dispersal by nutcrackers (probability > or = 0.7), and that this level of cone production can be met by forests with live whitebark pine basal area > 5.0 m2/ha. The risk of mutualism disruption is greatest in northern most Montana (USA), where three-year mean cone production and live basal area fell below predicted threshold levels. There, nutcracker occurrence, seed dispersal, and whitebark pine regeneration were the lowest of the three ecosystems. Managers can use these threshold values to differentiate between restoration sites requiring planting of rust-resistant seedlings and sites where nutcracker seed dispersal can be expected.

Growth form distribution and genetic relationships in tree clusters of Pinus flexilis, a bird-dispersed pine.

Seed dispersal by the Clark's nutcracker (Nucifraga columbiana Wilson) may markedly influence the growth form and genetic population structure of limber pine (Pinus flexilis James). The nutcracker buries clusters of seeds in subterranean caches; germination of clustered seeds often results in a growth form characterized by two or more genetically distinct trees with fused or contiguous trunks (tree clusters). The occurrence of a morphologically similar form, the multi-trunk tree (a single genet branched near the base), as well as the typical single-trunked tree, complicates the study of limber pine populations. We examined growth form distribution and genetic relationships in tree clusters in limber pine populations at four elevations (from 2585 m to 3460 m) in the Colorado Front Range. At three study areas, relative occurrence of limber pine growth forms, as well as that of associated pines, was examined by a point-centered quarter survey. From the four study areas, we collected foliage from each trunk from a total of 74 "clumps" (combined tree clusters and multi-trunk trees) in order to differentiate the two growth forms using starch gel protein electrophoresis. Tree "clumps" were significantly more common in limber pine than in ponderosa or lodgepole pine (P<0.010). Although single-trunk limber pine was the most common growth form, except at the highest elevation, both multi-trunk trees and tree clusters were present in each stand. Tree clusters were estimated to comprise about 20% of the tree sites in each limber pine stand; the estimated proportion of multi-trunk trees varied by site from 5% to 77%. Trees in clusters were related, on average, as half to full siblings (mean r=0.43), but were unrelated to trees in other clusters (mean r=0.01). Electrophoretic analysis suggests possible genetic differentiation in limber pine that may be the result of different selection pressures on the growth forms.

Seed dispersal by nutcrackers causes multi-trunk growth form in pines.

We present a unique example of a seed disperser determining the basic growth architecture of two species of plants. Clark's Nutcracker (Nucifraga columbiana) is the primary disperser for Pinus flexilis and P. albicaulis, caching clusters of seeds in the process. Both pine species often occur in a multi-trunk growth form at maturity. Electrophoretic analysis based on 2 to 4 gene loci revealed that two or more trunks were genetically distinct individuals in twenty of the twenty-five multi-trunk trees sampled. This supports the hypothesis that several mature individuals can arise from single caches.